Many diseases such as diabetes, hypertrophic cardiomyopathy, hypothyroidism and heart failure involve alterations in the contractile and regulatory proteins of the myocardium. Changes in protein isoforms due to disease or mutation could impair cardiac function during normal activation of the heart or during strenuous activity. The long term objective of our research is to understand the complex molecular interactions between actomyosin cross-bridges (CBs) and thin filament (TF) proteins that regulate cardiac contractile function. In skeletal muscle the CB transition rates that control force development and fiber shortening have been studied using caged compound techniques and by varying substrate and hydrolysis product conditions. The CB processes that are rate limiting for force development and shortening in myocardium are less well understood than in skeletal muscle, and may be different. Additionally, the regulation of cardiac contractions appears to be dependent on a complex interaction between TF proteins and CBs. Using transient kinetics of isolated proteins in solution and glycerinated cardiac muscle we will study the rates of CB steps that govern actomyosin interaction and the influence of TnC Ca/2+ binding kinetics, comparing these with skeletal muscle. To test this we will independently alter the kinetics of TF activation and CB transition rates to determine how each affects steady state force and stiffness, the rate of force development and the velocity of shortening. We will also study the influence of protein phosphorylation and contractile filament lattice spacing on these measurements. The information gained from these studies will be used to modify and extend existing models of CB chemomechanical transduction and the regulation of CBs by TF proteins. These models will be useful in describing the important differences between cardiac and skeletal muscle in the control of force and shortening and the changes that occur in cardiac myopathies.